Andrew G. McCubbin

Identification of the pollen determinant of S-RNase-mediated self-incompatibility

Many flowering plants have adopted self-incompatibility mechanisms to prevent inbreeding and promote out-crosses. In the Solanaceae, Rosaceae and Scrophulariaceae, two separate genes at the highly polymorphic S-locus control self-incompatibility interactions: the S-RNase gene encodes the pistil determinant and the previously unidentified S-gene encodes the pollen determinant. S-RNases interact with pollen S-allele products to inhibit the growth of self-pollen tubes in the style. Pollen-expressed F-box genes showing allelic sequence polymorphism have recently been identified near to the S-RNase gene in members of the Rosaceae and Scrophulariaceae; but until now have not been directly shown to encode the pollen determinant. Here we report the identification and characterization of PiSLF, an S-locus F-box gene of Petunia inflata (Solanaceae). We show that transformation of S1S1, S1S2 and S2S3 plants with the S2-allele of PiSLF causes breakdown of their pollen function in self-incompatibility. This breakdown of pollen function is consistent with ‘competitive interaction’, in which pollen carrying two different pollen S-alleles fails to function in self-incompatibility. We conclude that PiSLF encodes the pollen self-incompatibility determinant.

Calcium-Dependent Protein Kinase Isoforms in Petunia Have Distinct Functions in Pollen Tube Growth, Including Regulating Polarity

Calcium is a key regulator of pollen tube growth, but little is known concerning the downstream components of the signaling pathways involved. We identified two pollen-expressed calmodulin-like domain protein kinases from Petunia inflata, CALMODULIN-LIKE DOMAIN PROTEIN KINASE1 (Pi CDPK1) and Pi CDPK2. Transient overexpression or expression of catalytically modified Pi CDPK1 disrupted pollen tube growth polarity, whereas expression of Pi CDPK2 constructs inhibited tube growth but not polarity. Pi CDPK1 exhibited plasma membrane localization most likely mediated by acylation, and we present evidence that suggests this localization is critical to the biological function of this kinase. Pi CDPK2 substantially localized to as yet unidentified internal membrane compartments, and this localization was again, at least partially, mediated by acylation. In contrast with Pi CDPK1, altering the localization of Pi CDPK2 did not noticeably alter the effect of overexpressing this isoform on pollen tube growth. Ca2+ requirements for Pi CDPK1 activation correlated closely with Ca2+ concentrations measured in the growth zone at the pollen tube apex. Interestingly, loss of polarity associated with overexpression of Pi CDPK1 was associated with elevated cytosolic Ca2+ throughout the bulging tube tip, suggesting that Pi CDPK1 may participate in maintaining Ca2+ homeostasis. These results are discussed in relation to previous models for Ca2+ regulation of pollen tube growth.

Genetic mapping and molecular characterization of the self-incompatibility (S) locus in Petunia inflata

Gametophytic self-incompatibility (SI) possessed by the Solanaceae is controlled by a highly polymorphic locus called the S locus. The S locus contains two linked genes, S-RNase, which determines female specificity, and the as yet unidentified pollen S gene, which determines male specificity in SI interactions. To identify the pollen S gene of Petunia inflata, we had previously used mRNA differential display and subtractive hybridization to identify 13 pollen-expressed genes that showed S-haplotype-specific RFLP. Here, we carried out recombination analysis of 1205 F2 plants to determine the genetic distance between each of these S-linked genes and S-RNase. Recombination was observed between four of the genes (3.16, G211, G212, and G221) and S-RNase, whereas no recombination was observed for the other nine genes (3.2, 3.15, A113, A134, A181, A301, G261, X9, and X11). A genetic map of the S locus was constructed, with 3.16 and G221 delimiting the outer limits. None of the observed crossovers disrupted SI, suggesting that all the genes required for SI are contained in the chromosomal region defined by 3.16 and G221. These results and our preliminary chromosome walking results suggest that the S locus is a huge multi-gene complex. Allelic sequence diversity of G221 and 3.16, as well as of 3.2, 3.15, A113, A134 and G261, was determined by comparing two or three alleles of their cDNA and/or genomic sequences. In contrast to S-RNase, all these genes showed very low degrees of allelic sequence diversity in the coding regions, introns, and flanking regions.

S-RNases and sexual incompatibility: structure, functions, and evolutionary perspectives

S-RNase-based gametophytic self-incompatibility appears to be the most phylogenetically widespread form of self-incompatibility found in the angiosperms, having been reported in the Solanaceae, Scrophulariaceae, and Rosaceae. This intraspecific breeding barrier is controlled by a single genetic locus termed S. Rejection of self-pollen has been shown to be mediated in the pistil by a highly polymorphic series of ribonucleases, but as yet the pollen component of this recognition system has not been identified. Here we review our present knowledge concerning the structure, functions, and evolution of S-RNases and the S-loci in which they reside. In addition we present two new phylogenetic analyses of S-RNases which suggest that (1). sequence variability between S-alleles is spread across the whole gene and is not as clustered as is generally believed and (2). there is evidence of recombination and/or diversifying selection in two distinct regions of S-RNases. The implications of these findings are discussed.

Identification of genes showing differential expression between morphs in developing flowers of Primula vulgaris

Heteromorphic self-incompatibility systems provide an excellent model for studying both intraspecific breeding barriers and the regulation of floral organ size and positioning, yet at the molecular level, they are almost completely uncharacterized. In this study, a subtracted cDNA library was generated from developing and mature floral tissues of the thrum morph of Primula vulgaris, subtracted with the same tissues from the pin morph. Differential screening and reverse transcriptase-polymerase chain reaction analysis identified 11 classes of cDNA that were differentially expressed between developing floral morphs. A number of these classes have significant homology to members of gene families implicated in plant development, including rapid alkalinization factors, DExH box RNA helicases, SKS multi-copper oxidases, and AtCHX ion-transporter families, consistent with their potential involvement in the regulation of floral heteromorphy. None of the cDNAs identified appear to be linked to the PrimulaS-locus suggesting that they are not components of the S-locus itself, and are more likely downstream components of developmental pathways leading to floral heteromorphy.

The emerging complexity of self-incompatibility (S-) loci

Abstract A complex picture of S-loci is beginning to emerge from recent studies of the S-locus of RNase-based gametophytic self-incompatibility displayed by the Rosaceae, Solanaceae, and Scrophulariaceae, and of the S-locus of the type of sporophytic self-incompatibility displayed by the Brassicaceae. It now appears that not only do these S-loci contain two separate genes, one controlling pollen function and the other controlling pistil function in self-incompatibility interactions, but also many other genes whose functions are largely unknown. The implications of these recent findings for the study of the mechanisms of self-incompatibililty interactions and evolution of the self-incompatibility systems are discussed.

The pollen-specific R-SNARE/longin PiVAMP726 mediates fusion of endo- and exocytic compartments in pollen tube tip growth

The growing pollen tube apex is dedicated to balancing exo- and endocytic processes to form a rapidly extending tube. As perturbation of either tends to cause a morphological phenotype, this system provides tractable model for studying these processes. Vesicle-associated membrane protein 7s (VAMP7s) are members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family that mediate cognate membrane fusion but their role in pollen tube growth has not been investigated. This manuscript identifies PiVAMP726 of Petunia inflata as a pollen-specific VAMP7 that localizes to the inverted cone of transport vesicles at the pollen tube tip. The endocytic marker FM4-64 was found to colocalize with yellow fluorescent protein (YFP)-PiVAMP726, which is consistent with PiVAMP726 containing an amino-acid motif implicated in endosomal localization, At high overexpression levels, YFP- PiVAMP726 inhibited growth and caused the formation of novel membrane compartments within the pollen tube tip. Functional dissection of PiVAMP726 implicated the N-terminal longin domain in negative regulation of the SNARE activity, but not localization of PiVAMP726. Expression of the constitutively active C-terminal SNARE domain alone, in pollen tubes, generated similar phenotypes to the full-length protein, but the truncated domain was more potent than the wild-type protein at both inhibiting growth and forming the novel membrane compartments. Both endo- and exocytic markers localized to these compartments in addition to YFP-PiVAMP726, leading to the speculation that PiVAMP726 might be involved in the recycling of endocytic vesicles in tip growth.

Sequencing the genomic regions flanking S-linked PvGLO sequences confirms the presence of two GLO loci, one of which lies adjacent to the style-length determinant gene CYP734A50

Key messagePrimula vulgaris contains twoGLOBOSA loci, one located adjacent to the style length determinant geneCYP734A50 which lies within theS-locus.AbstractUsing a combination of BAC walking and PacBio sequencing, we have sequenced two substantial genomic contigs in and around the S-locus of Primula vulgaris. Using these data, we were able to demonstrate that two alleles of PvGloP as well as PvGloT can be present in the genome of a single plant, providing empirical evidence that these two forms of the MADS-box gene GLOBOSA are separate loci and not allelic as previously reported. We propose they should be renamed PvGLO1 and PvGLO2. BAC contigs extending from each GLOBOSA locus were identified and fully sequenced. No homologous genes were found between the contigs other than the GLOBOSA genes themselves, consistent with their identity as separate loci. Exons of the recently identified style-length determinant gene CYP734A50 were identified on one end of the contig containing PvGLO2 and these genes are adjacent in the genome, suggesting that PvGLO2 lies either within or at least very close to the S-locus. Current evidence suggests that both CYP734A50 and GLO2 are specific to the S-morph mating type and are hemizygous rather than heterozygous in the Primula genome. This finding contrasts classical models of the HSI locus, which propose that components of the S-locus are allelic, suggesting that these models may need to be reconsidered.

A social stigma: Plant biology

Self-incompatibility is a strategy used in some bisexual flowering plants to avoid inbreeding; for that purpose, the female pistil tissue needs to distinguish between pollen from the same plant and that from other plants. Research withBrassica species has unveiled an element of the genetic mechanism of self-incompatibility — the so-called SRKgene controls the pistils ability to recognize and reject the same plants pollen.

PiSCP1 and PiCDPK2 Localize to Peroxisomes and Are Involved in Pollen Tube Growth in Petunia Inflata

Petunia inflata small CDPK-interacting protein 1 (PiSCP1) was identified as a pollen expressed PiCDPK1 interacting protein using the yeast two hybrid system and the interaction confirmed using pull-down and phosphorylation assays. PiSCP1 is pollen specific and shares amino acid homology with uncharacterized proteins from diverse species of higher plants, but no protein of known function. Expression of PiSCP1-GFP in vivo inhibited pollen tube growth and was shown to localize to peroxisomes in growing pollen tubes. As PiCDPK1 is plasma membrane localized, we investigated the localization of a second isoform, PiCDPK2, and show that it co-localizes to peroxisomes with PiSCP1 and that the two proteins interact in the yeast 2 hybrid interaction assay, suggesting that interaction with the latter CDPK isoform is likely the one of biological relevance. Both PiCDPK2 and PiSCP1 affect pollen tube growth, presumably by mediating peroxisome function, however how they do so is currently not clear.